We examined the neuro/axono-protective potential of a novel poly (ADP-ribose) polymerase (PARP) inhibitor L-2286 in a rat impact acceleration brain injury model. Male Wistar rats (n = 70) weighing 300–350 grams were used to determine the most effective intracerebroventricular (i.c.v.) dose of L-2286 administered 30 min after injury, and to test the neuroprotective effect at two time points . The neuroprotective effect of L-2286 was tested using immunohistochemical (amyloid precursor protein and mid-sized mouse anti-neurofilament clone RMO-14.9 antibody) and behavioral tests . At both time-points, a 100 μg/rat dose of i.c.v. L-2286 significantly (p < 0.05) reduced the density of damaged axons in the corticospinal tract and medial longitudinal fascicle compared to controls. In the behavioral tests, treatment 30 min post-injury improved motor function, while the level of anxiety was reduced in both treatment protocols. Although 50 μg/rat of i.c.v. L-2286 reduced the density of damaged profiles, a significant difference could not be demonstrated. Ultimately, 100 μg/rat of i.c.v. L-2286 significantly reduced the APP-IR axons in CSpT .2.2.The immunohistochemical examination, and the following digital data assessment and statistical analysis, revealed that 100 μg/rat of i.c.v. L-2286 in both treatment paradigms significantly reduced the density of APP and mid-sized mouse anti-neurofilament clone RMO-14.9 (RMO-14) IR axons after head injury.2) in both the CSpT and medial longitudinal fascicle (MLF) to a similar degree compared to vehicle-treated animals. Both treatment time points were effective in terms of significantly reducing the density of RMO-14-IR axons in CSpT and MLF compared to vehicle-treated animals. However, immediate post-injury treatment seems to be more effective, since a significant difference was observed in the density of damaged axons in both CSpT and MLF compared to 30 min post injury  during the seven-day period. The group treated 30 min post-injury reached the maximal accessible score on the fourth day. The motor performance of this group was significantly improved from the second examined time-point (one hour) up to the last time-point (seventh day) compared to the vehicle-treated group .2.4.The open-field test measures altered motor performance and the level of anxiety. Results of the test showed that there were significantly fewer crossing and grooming activities in the vehicle-treated group than in the sham-injured group. Immediate post-injury treatment with L-2286 did not significantly improve any of the three examined parameters compared to vehicle-treated animals. However, L-2286 treatment 30 min post-injury significantly improved the number of grooming activities compared to the vehicle-treated group, but without any effect on the another two parameters (crossing and rearing) .2.5.Results of the elevated plus-maze test indicate that the vehicle-treated animals suffered from anxiety after injury. A statistically significant difference between the sham-injured and vehicle-treated groups was detected in the number of head dips in the open arm, time in the open arm, time in the closed arm, and the amount of grooming in the closed arm. The anxiety level was significantly lower in the L-2286-treated group than in the vehicle treated animals, since rats spent significantly more time in the “unsafe” open arm than in the “safe” closed arm. Interestingly, the group treated immediately post-injury not only spent significantly more time in the open arm than the vehicle treated group, but also the sham-injured group, a finding not observed in rats treated 30 min post-injury. The vehicle-treated and the latter group significantly differed only in the parameters of head dips in the open arm, time in the open arm and time in the closed arm –10.2.6.In our study, we demonstrated that the novel PARP inhibitor L-2286 is capable of preventing both morphological  and neurobehavioral sequelae associated with severe TBI evoked in a rat IA head injury model.The first stage of the study determined the effective dose of this new drug in a post-injury administration paradigm using immunohistochemical analysis of APP accumulation in CSpT. This assay was chosen as APP is recognized as the primary  marker of diffuse axonal injury (DAI) evoked by TBI. Intracerebroventricular treatment with different doses of L-2286 30 min post-injury proved that 100 μg/rat is the most effective dose of those applied .In the second phase of the experiment, APP and RMO-14 immunohistochemistry was used to further elucidate the efficiency of 100 μg/rat of i.c.v. L-2286 administered at time points after TBI (immediately and 30 min post-injury). Both time points were effective in terms of significantly reducing the densities of damaged axons in CSpT and MLF. It should be noted, however, that in the case of RMO-14-IR axons, immediate post-injury treatment appeared to be far more effective, although this difference was not observed in APP-IR axons.i.e., the formation of axonal bulbs (predominantly detected by APP-IR) and cytoskeletal alterations reflected in neurofilament compaction (RMO-14-IR) [To the best of our knowledge, this study is the first attempt to assess the efficacy of PARP-inhibition on DAI. Studies have thus far aimed at analyzing poly(ADP-ribose) immunohistochemistry  or changO-14-IR) ,12, whicOur qualitative and quantitative immunohistochemical results corroborate earlier observations that show the complexity of TAI pathogenesis. Specifically, we demonstrated that RMO-14 and APP-IR axon populations display different reactions to the same neuroprotective intervention, a finding well explained in the aforementioned studies.Based on the finding that there is a strong decrease in the density of damaged IR axonal profiles, we assessed the functional efficacy of the therapeutic strategies applied. Many articles have demonstrated that Marmarou’s impact acceleration model impairs cognitive and motor functions in rodents –15. The in vitro and in vivo, with significant neuroprotective properties following ischemia-reperfusion in rats. This suggests that this compound or its derivative could not only be an important tool to investigate the physiological role of PARP in neurodegenerative pathways, but also a therapeutic option for stroke and neurodegenerative disease.Application of PARP-inhibitors may be useful in the treatment of numerous neurodegenerative diseases. The brain is particularly susceptible to radical-mediated neuronal damage due to high levels of oxygen consumption, unsaturated fatty acids, and iron stores, combined with low antioxidant resources. Oxidative stress is a critical step in neuronal degeneration –21. Iwas+) depletion contribute to cell death, its absence or inhibition may prevent cell death associated with the apoptotic cascade and energy depletion. There is solid evidence for the beneficial effects of energy conservation (via NAD+ conservation) with PARP-inhibition in ischemic insults to the brain [Provided that PARP activation and nicotinamide-adenine-dinucleutide  brain injury in mice. They found improved Morris water-maze performance with no difference in contusion volume, hippocampal neuron survival, or motor performance. Examining the effect of i.p. INO-1001 30 min before and three times daily for three days after fluid percussion injury in rats, Besson and coworkers [PARP-inhibitors have been shown to improve neurological outcome in models of head injury ,6,8,10. oworkers  demonstroworkers . In hypooworkers .As maintenance of energy homeostasis in the prevention of DAI has been proven to be of ample importance –33, it s3.ad libitum for two weeks prior to the study. The experiments were carried out in accordance with regulations of the Hungarian Animal Care Committee (BA02/2000-26/2001).Sixty-eight adult male Wistar rats weighing 300–350 g  were used for the experiments. Animals were housed in single cages under controlled environmental conditions  with food and water 3.1.2O and 30% O2. After endotracheal intubation, rats were ventilated with 1.5% isoflurane in 70% N2O and 30% O2 . Arterial oxygen saturation was measured via pulse oximetry (Nonin 8600V), rectal and temporal temperatures were monitored and maintained at 37 °C with a feedback-controlled heating pad (FHC BOWDOINHAM ME 04008 USA Temperature Control). A midline incision was made to expose the skull from the bregma to the lambda sutures. A stainless steel disc (10 mm in diameter and 3 mm thickness) was fixed centrally between the lambda and bregma sutures using cyanoacrylate. A 450-g weight was dropped from 2 m onto the stainless disc fixed to the rat’s skull. After impact, the metal disc was removed and the animals were monitored during the recovery of spontaneous respiration. Sham animals were prepared for injury in the same fashion, but were not injured.The injury protocol was the same in all experiments. All rats were first anesthetized in a bell jar for 5 min with 4% isoflurane  in 70% N3.2.To establish the dose-response curve for L-2286, each rat (n = 5 in each group) was given a single bolus of 10, 50 or 100 μg/rat of L-2286 dissolved in 5 μL of physiological saline. The drug was administered i.c.v. 30 min after severe head injury through a craniotomy 1 mm posterior, 1.5 mm lateral and 3.5 mm deep relative to bregma. The vehicle group (n = 5) received i.c.v. 5 μL/rat of physiological saline. Two hours post-injury, animals were sacrificed for immunohistological assessment to determine the dose-response curve in terms of a reduction in the density of damaged IR axons in the corticospinal tract.3.3.Rats (n = 24) received a single 100 μg/rat bolus of i.c.v. L-2286 dissolved in 5 μL of physiological saline, while vehicle-treated animals were treated with i.c.v. 5 μL/rat of physiological saline. L-2286 was administered either 0 min (n = 12) or 30 min (n = 12) after injury. Animals for histological analysis (n = 24) were sacrificed 2 h after brain injury. The remaining 30 animals survived 9 days post-injury and underwent behavioral tests each day.3.4.Two hours after injury, rats were sacrificed with i.p. sodium pentobarbital and transcardially perfused with 4% paraformaldehyde in Millonig’s buffer. Brains were removed from the skull and immersed in the same fixative overnight (16–18 h). Five mm-wide blocks of the brain were cut out using a sagittal brain-blocking device (Braintree Scientific Inc.), which include the regions extending from the interpeduncular fossa to the first cervical segment and were immersed in the same fixative for 24 h. Blocks were sectioned with a Vibratome Series 1500  at a thickness of 30 μm and collected in 0.1 M phosphate buffer in a semi-serial fashion. Adjacent sections were then processed for immunohistochemical detection of damaged axonal profiles. Impaired axoplasmatic transport, long associated with TAI, was assessed by immunohistochemical detection of APP ,33–38. A3.5.2 grid was superimposed over the CSpT and adjacent MLF region, and all damaged axonal profiles within the grid were counted and expressed as density of APP or RMO-14-IR axons (IR-axons/mm2).APP or RMO-14 IR axonal profiles were examined using a NIKON light microscope interfaced with a SPOT RT digital camera and a computer-assisted image analysis system (IMAGE-PRO PLUS v5.0.1) in a blinded fashion. The area of CSpT and MLF was analyzed. Six sections containing both CSpT and MLF regions from each animal were captured and digitized at a magnification of 10×. A 180,000 μm3.6.Behavioral tests were conducted one day before  and from 1 h to 9 days after injury. The trials were scored blind by a single observer. The behavioral tests were done during the light phase. Between each trial, all apparatus were cleaned with a 75% ethanol solution.3.6.1.Before injury, animals were given repeated training until capable of balancing on the beam for three consecutive trials of 60 sec each. Rats were placed at the center of a 1.5-cm-wide square wooden bridge that was suspended 60 cm above a foam pillow. All animal groups were tested after the injury  for 7 days (1 h and 1–7 days after injury) and scored from 1–6 on beam balance . All tri3.6.2.The open-field test was used to measure spontaneous locomotor activity. The open-field is a box with a 70 × 70 cm floor and a height of 50 cm. On the eighth day post–injury, rats were brought into the experimental room. They remained in their transfer cages for 30 min, after which each subject was placed into the center of the open-field box. Crossing, rearing and grooming were recorded with a video camera during the 5-minute test.3.6.3.The elevated plus-maze was used to measure anxiogenic behavior. The maze consisted of 4 intersecting arms and was constructed from varnished wood. The maze was elevated 1 m above the floor. The arms of the maze were 45 cm long and 10 cm wide; the closed arms had walls on 3 sides that were 30 cm high, and the open arms did not have walls on any side. Nine days after the trauma, rats were placed one-by-one in the center of the maze facing one of the closed arms. An observer, who was seated 1.5 m from the apparatus, recorded with a video camera the number of head dips into the open arms, entries with the first limbs into the open arms, grooming in both the closed and open arms and the time in closed or open arms.3.6.4.2) between L-2286- and vehicle-treated animals. For beam-balance, the scores of each group were totaled and averaged daily. ANOVA with Dunn’s post hoc tests were used to compare the scores of experimental groups. For open-field, the number of crossings, rearings and the time spent in central and peripheral zones during the 5-minute test were analyzed. For elevated plus-maze, the number of head dips into the open arms, entries with the first limbs into the open arms, grooming in both closed and open arms and the time spent in closed or open arms were analyzed. Results of open-field and elevated plus-maze were analyzed with ANOVA and Dunnett’s post hoc tests. Differences were considered significant with a p value of 0.05. Statistical analysis was performed using Graph Pad Instat software.ANOVA with Dunnett’s post hoc test was used to compare the density of immunopositive axonal-profiles (expressed as mean number/mm4.Our results strongly suggest the neuroprotective effect of PARP inhibition; the heterogeneity of the results mandates further, detailed experiments that should shed light on the exact subcellular pathomechanism of this PARP inhibition. Specifically, we should elucidate the role of diffuse neural injury not assessed in the present experiments that may lay behind the observed motor-behavioral benefit provided by L-2286. To this end, further studies in different models, primarily in fluid percussion head injury, are planned, while also assessing ultrastructural details, particularly mitochondrial integrity in the axonal bundles that are prone to diffuse TBI.


Aedes aegypti L. (Diptera: Culicidae) were mounted on perfusion pipettes and bathed in high buffer mosquito saline. With low buffer perfusion saline, containing m-cresol purple, transepithelial voltage was monitored and luminal alkalinization became visible through color changes of m-cresol purple after perfusion stop. Lumen negative voltage and alkalinization depended on metabolic energy and were stimulated in the presence of serotonin (0.2 µmol l-1). In some experiments a pH microelectrode in the lumen recorded pH values up to 10 within minutes after perfusion stop. The V-ATPase inhibitor concanamycin (50 µmol l-1) on the hemolymph side almost abolished Vte and inhibited luminal alkalinization. The carbonic anhydrase inhibitor, methazolamide (50 µmol l-1), on either the luminal or hemolymph-side, or the inhibitor of anion transport, DIDS (1 mmol l-1) on the luminal side, had no effect on Vte or alkalinization. Cl- substitution in the lumen or on both sides of the tissue affected Vte, but the color change of m-cresol purple was unchanged from control conditions. Hemolymph-side Na+ substitution or addition of the Na+/H+ exchange inhibitor, amiloride (200 µmol l-1), reduced Vte and luminal alkalinization. Luminal amiloride (200 µmol l-1) was without effects on Vte or alkalinization. High K+ (60 mmol l-1) in the lumen reduced Vte without affecting alkalinization. These results indicate that strong luminal alkalinization in isolated and perfused anterior midgut of larval A. aegypti depends on basolateral V-ATPase, but is apparently independent of carbonic anhydrase, apical Cl-/HCO3- exchange or apical K+/2H+ antiport.In the present study, isolated midguts of larval  Ion gradients indicative for HCl efflux from the tissue to the hemolymph were abolished or reduced after application of the V-ATPase inhibitor, bafilomycin, inhibitors ofcarbonic anhydrase or DIDS. These results were summarized in a transport model where basal V-ATPase energizes apical Cl-/HCO3- exchangers. The intracellular substrates of these transporters were proposed to be rapidly supplied through the action of carbonic anhydrase. Cl- ions that enter the cells from the lumen via the exchangers were proposed to exit to the hemolymph through basal Cl- channels. It was recognized, however, that this HCO3- secretion alone can only explain luminal alkalinization up to pH values of about 8.5, and that alkalinization to higher pH values would need additional active and transepithelial absorption of H+ (or secretion of strong base).Boudko et al. , b introte) of isolated and perfused midgut segments. The initially very high Vte of anterior (lumen negative) and posterior (lumen positive) midgut segments rapidly declined after mounting and were only partly restored after addition of hemolymph-like, submicromolar concentrations of serotonin. In a subsequent study of the isolated anterior midgut (te generated by the anterior midgut reflects HCO3- secretion via one cell type and H+ absorption (or strong base secretion) via the other cell type. However, isolation and mounting of the tissue deactivates both cell populations and serotonin only reactivates the HCO3- secreting cells. Onken et al.  eggs were provided by Dr. Marc Klowden  from a continuously maintained colony. Eggs were hatched and larvae were maintained in a 1:1 mixture of tap water and deionized water at 26 °C and on a 16:8 L:D photoperiod. The water was replaced each morning, and the larvae were fed with ground Tetramin flakes . Fed fourth-instar larvae were used in all experiments.Aedes hemolymph composition (-1): NaCl, 42.5; KCl, 3.0; MgCl2, 0.6; CaCl2, 5.0; NaHCO3, 5.0; succinic acid, 5.0, malic acid, 5.0; L-proline, 5.0; L-glutamine, 9.1; L-histidine, 8.7; L-arginine, 3.3; dextrose, 10.0; Hepes, 25. The pH was adjusted to 7.0 with NaOH. In Na+-free saline, NaCl was substituted with N-methylglucamine. Instead of 5 mmol l-1 NaHCO3, this saline contained 3 mmol l-1 KHCO3 (no KCl). The pH was adjusted with HCl. In Cl--free saline, gluconates  or sulfate (Mg2+) substituted for the chlorides. The pH was adjusted with NaOH. The salines to perfuse the lumen of the tissue were identical to the above salines, but contained 0.04 % m-cresol purple  and reduced buffer (0.25 mmol l-1 Hepes). In the high K+ saline the K+ concentration was increased to 60 mmol l-1 by replacing NaHCO3 with KHCO3 and part (35 mmol l-1) of NaCl with KCl (titration with KOH 20 mmol l-1). The above components were purchased from Sigma , Fisher Scientific (www1.fishersci.com) or Mallinckrodt . Serotonin (Sigma) and dinitrophenol  were directly dissolved in the saline. Amiloride (Sigma) was added to the saline from an aqueous stock solution of 10 mmol l-1. Concanamycin , methazolamide and 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid  were added from stock solutions in dimethylsulfoxide . The primary solvent alone had no effect on voltage or alkalinization at the concentrations present in the experiments.The basic NaCl saline used to perfuse the bath (hemolymph-side of the epithelium) was based on larval position , b and cManufacture of perfusion pipettes as well as preparation, mounting and perfusion of anterior midguts was almost identical to those outlined in detail before ,b and wiwww.brinkmann.com) until the tip of the pipette recorded the typical, high, lumen negative transepithelial voltage (Vte). The preparations were tied in place with a fine human hair. In order to keep the preparation in the focus of the binocular microscope, the open anterior end of the midgut preparation was slipped onto a glass rod manufactured from a glass capillary pipette  and held by a second micromanipulator. The bath (volume 100 µl) was gravity perfused (rate 15–30 ml h-1) with oxygenated salines. The perfusion pipette contained a polyethylene tubing  and was closed with a syringe needle. The syringe needle and the tubing were connected to two push-pull multi-speed syringe pumps , allowing fast changes of the luminal perfusion (rate 30 µl h-1) between two different solutions. The transepithelial voltage (Vte) was measured exactly as described before . Color changes were documented with a digital camera  before and at 5 minute time intervals after perfusion stop. All photographs were identically edited  with iPhoto Express . In order to estimate the pH, polyethylene tubing with the approximate dimensions of the anterior midgut  was inserted into the bath and perfused with NaCl saline of different pH, containing 0.04 % m-cresol purple. The colors of these solutions were documented as described above and are shown in pK1 ∼ 1.65, pK2 = 9.20) was used instead of m-cresol purple. In some experiments pH-sensitive microelectrodes were prepared . Differences between groups were tested, using Student's t-test. Significance was assumed at te) of -12 ± 1 mV  was measured. After application of 0.2 µmol l-1 serotonin the mean lumen negative Vte increased to a more negative value of -45 ± 3 mV . These voltages were very similar to the results obtained previously with the same tissue  and alkalinization was markedly reduced . Hemolymph-side solution: high buffer mosquito saline with serotonin (0.2 µmol l-1). The posterior part of the preparation is tied with a human hair onto a perfusion pipette (left) and a glass rod inserted into the lumen (right) maintains the tissue in the focus of the microscope. Luminal perfusion was stopped at the beginning of the video. The color change of m-cresol purple that reflects alkalinization (from yellow to purple) begins at the anterior end of the midgut (right) and continues towards more posterior regions of the anterior midgut (left). The peristaltic muscular activity can also be easily observed  shows a typical experiment with an isolated and perfused anterior midgut of larval rved cf. . At 2:40+ and Cl- at the hemolymph-side surface of the anterior midgut of semiintact larvae that are indicative for HCl flux from the epithelial cells to the hemolymph (-1) on Vte and alkalinization in isolated and perfused anterior midgut preparations were monitored. Applying the drug to the hemolymph-side bath for 10 to 20 minutes had no significant effect on Vte (P > 0.05), and alkalinization was equally observed in the presence and in the absence of methazolamide  and alkalinization were observed when the lumen of the isolated anterior midgut was perfused for 10 to 15 minutes with methazolamide  markedly reduced alkalinization in the anterior midgut of intact larvae . Moreoveemolymph . On the emolymph . In the + and Cl- gradients at the surface of the exposed anterior midgut of semiintact larvae. Onken et al. , but this manipulation did not significantly affect Vte (P > 0.05) and alkalinization was observed as in the controls before addition of the drug . Alkalinization was still observed as in the control before substitution of luminal Cl-. Subsequent additional substitution of Cl- in the hemolymph-side bath for another 10 minutes significantly reduced the lumen negative voltage to -18 ± 5 mV , but alkalinization was observed even under these conditions , was used to evaluate the capacity of alkalinization in addition to the effects on Vte. As in the previous study, hemolymph-side amiloride significantly reduced Vte from -52 ± 11 to -19 ± 4 mV  was needed to abolish K+/2H+ antiport in membrane vesicles from M. sexta midgut  to reduce or abolish the transapical K+ gradient. This manipulation should at least reduce transapical K+/2H+ antiport. In five experiments high luminal K+ significantly reduced Vte by approximately 20 % from -42 ± 4 to -33 ± 3 mV  after an average exposure time of 8 minutes, but the color change of mcresol purple was the same as observed under control conditions  at a higher pH value than m-cresol purple. In three experiments isolated anterior midgut preparations were perfused with low buffer saline containing 0.04 % thymol blue. After perfusion stop, color changes from yellow to blue were observed, confirming the above estimate that the alkalinization in isolated anterior midgut preparations reaches pH values above 9. In three experiments a pH-sensitive microelectrode was inserted into the lumen through the open end of the preparation. After stopping the luminal perfusion, Vte decreased, m-cresol purple changed color to pink, and the recorded pH rapidly increased. In one experiment  were observed to generate a midgut pH of up to 12  dramatically dropped to less negative values after mounting of the isolated tissue , the anterior midgut of larval A. aegypti loses at least a significant part of its capacity for strong alkalinization after isolation and mounting.One problem that impedes analyses of the mechanisms of strong luminal alkalinization in isolated insect midgut regions is that up to 12 , the pH up to 12 . Indeed,M. sexta . However necessary for strong alkalinization. Other attempts to restore the in vitro transepithelial potential difference with a number of peptide hormones  at high hemolymph Cl- . In any case, this work has established that serotonin alone is sufficient to reestablish alkalinization similar to that seen in the intact animal.Now that it is known that addition of serotonin is sufficient to re-establish strong alkalinization, the question of the discrepancy between the initially high V+ pumps  is essential for strong alkalinization in the midgut of larval mosquitoes . Indeed,M. sexta , but CA M. sexta . In the mide see  nor methmide see , 6 signiline see , 6. We t aegypti . Moreove aegypti . On the  aegypti . As in t aegypti  cell sur aegypti  and a su aegypti , 6 shows3- secretion alone cannot generate a strong alkalinization above pH values of 8.5, and therefore cannot offer a complete explanation for alkalinization. However, evidence for HCO3- secretion is provided by in vitro measurements: from the HCO3-/CO32- concentrations measured in the hemolymph and in the anterior midgut lumen of larval A. aegypti  was added first to the hemolymph-side bath and then introduced into the lumen by changing the pump from infusion to withdrawal. In the present study, a higher concentration of DIDS (1 mmol l-1) directly infused into the lumen did not at all affect Vte. Because changing the pump direction can cause voltage artifacts  and obtained the same result as in the previous paper. Consequently, we conclude that a different sequence of Cl- substitution  results in different responses of Vte. The differences between the results in the present and previous study are difficult to interprete and need to be addressed in the future.In a previous study, we reported somewhat different observations on the effects of DIDS and Cln et al.  measuredacts cf.  this metn et al.  also foue values . In the + markedly reduced lumen negative Vte to less negative values and it was proposed that a part of active, transepithelial base secretion/acid absorption proceeds via basolateral Na+/H+ exchangers and apical Na+/2-3HCO3- symporters. In the present study . Therefore, alternative interpretations for the effects of hemolymph-side amiloride and Na+ substitution must be considered. In addition to interfering with Na+/H+ exchange, amiloride and Na+ -free saline also inhibit Na+/Ca2+ exchangers  can be used to estimate a flux of 1–2 µmol cm-2 h-1. Indeed, this value is markedly smaller than the fluxes obtained with isolated midgut preparations of larval M. sexta, which were considered much lower than in vivo. However, it has to be considered that the ratio of epithelial surface and midgut volume is much larger in the small midgut of larval mosquitoes. In other words, in larval mosquitoes a much smaller surface-specific H+ absorption is necessary to increase the pH in the much smaller midgut volume.In summary, the present study shows that the isolated and perfused anterior midgut of larval odes see  confirme3- secretion, but they indicate that this process is not a prerequisite for strong alkalinization. Of course, on first sight our results seem to be disappointing. On the other hand, the observation that an in vitro preparation maintains its capacity for strong midgut alkalinization is an essential breakthrough. The experimental advantages of isolated epithelia for the analysis of strong alkalinization in an insect midgut suggest that understanding the underlying mechanisms can be achieved in the near future.The results of the present study indicate that, with the exception of V-ATPase involvement, all previous proposals about the mechanisms of strong alkalinization in larval mosquitoes seem to not apply. Indeed, our results do not exclude the presence of active HCO


The development of BM between the two treatment groups was significantly different . Patients who had received trastuzumab had longer times to BM compared with patients who were not treated with trastuzumab . Time to death (TTD) from BM was significantly longer in the post-T group than in the pre-T group . Extracranial disease control at the time of BM, 12 months or more of progression-free survival of extracranial disease and treatment with lapatinib were independent prognostic factors for TTD from BM.In patients with human epidermal growth factor receptor-2 (HER2)-overexpressing breast cancer, treatment with trastuzumab has been shown to markedly improve the outcome. We investigated the role of trastuzumab on brain metastasis (BM) in HER2-positive breast cancer patients. From 1999 to 2006, 251 patients were treated with palliative chemotherapy for HER2-positive metastatic breast cancer at Samsung Medical Center. The medical records of these patients were analysed to study the effects of trastuzumab on BM prevalence and outcomes. Patients were grouped according to trastuzumab therapy: pre-T (no trastuzumab therapy)  There has been a steady decline in the death rate from breast cancer worldwide since 1990. The development of new treatments, such as a human epidermal growth factor receptor-2 (HER2)-directed monoclonal antibody, has offered new hope to women with both early and advanced breast cancer whose tumours overexpress HER2. However, despite these clinical advances, as well as advances in our understanding of the related biology, breast cancer is still the main cause of cancer morbidity and mortality in women in most countries . In HER2Central nervous system (CNS) metastases, including parenchymal and leptomeningeal metastases, are the most common causes of malignant disease in the brain. Historically, the lack of durability for CNS control was not a problem for most patients because BMs occurred late in the course of illness, and progression in non-CNS was the dominant cause of morbidity and mortality , in terms of its biological behaviour of HER2.Secondly, we characterised the prevalence and predictors of CNS metastasis among patients with HER2-overexpressing MBC receiving trastuzumab-based therapy.in situ hybridisation (FISH). Grade 3 of the HER2 by immunohistochemical (IHC) staining was defined as a positive result for HER2, and amplification of the HER2 was confirmed by FISH if HER2 was rated 2+ by IHC. The pre-trastuzumab period (pre-T) was from 1999 to 2002. Most of the patients in this period did not receive trastuzumab because it was unavailable or not reimbursed by the Korean medical insurance system for clinical use. We used the patients in this period as the ‘historical control’. The post-trastuzumab period (post-T) was from 2003 to 2006. Trastuzumab was available and reimbursed by the insurance system in Korea for HER2-overexpressing breast cancer patients during this time period.We conducted a retrospective analysis of medical records from patients with histologically confirmed metastatic or recurrent breast cancer who received palliative chemotherapy at Samsung Medical Center from January 1999 to December 2006. All pathological specimens were reviewed by two experienced pathologists. HER2 status was evaluated using an antibody  and fluorescence Treatment modalities for BMs, either single or combined modalities, included whole brain radiation therapy (WBRT), surgical resection, stereotactic radiosurgery (SRS), including gamma-knife surgery (GKS) and systemic treatments such as chemotherapy and endocrine therapy. GKS was repeated for symptomatic control of metastatic brain lesion(s) if possible, with regular brain MRI follow-ups. Our study protocol was approved by the institutional review board of Samsung Medical Center.P-value <0.05 was considered significant. The differences in characteristics between pre-T and post-T were estimated by the χ2 test or Fisher's exact test. A binary logistic regression analysis was used for the multivariate analysis of each potential prognostic variable for development. A Cox proportional hazards regression model was used to assess the effect of each potential prognostic variable on TTD and OS. All potential prognostic variables were included in the model, and variables were removed from this model one at a time in a backward selection process using the likelihood ratio and a significance level of 0.05.The distant metastasis-free survival (DFS) was defined from the date of breast cancer diagnosis to the date of documentation of distant metastasis. The time to BM (TTBM) was defined from the date of distant metastasis to the date of BM. The time to death (TTD) from BM was defined from the date of BM to death or the last follow-up day. The overall survival (OS) was measured from the first day of treatment for MBC to the date of death or to the last follow-up day. The progression-free survival (PFS) of extracranial disease was defined from the first day of last chemotherapy regimen that had been administered prior to BM, to the date of progression of systemic disease irrespective of development or progression of CNS metastases. A switch in chemotherapy after development or progression of BM was allowed, and was not regarded as progression of extracranial disease. The brain PFS was defined from the last date of treatment for CNS metastasis to the date of progression of any CNS sites. Extracranial disease control was defined as systemic disease status, except in the CNS, with complete response (CR), partial response (PR) or stable disease (SD) at the time of BM. ‘Only BM’ was defined as absence of evidence of other distant metastasis except CNS. ‘First metastasis in brain’ was defined as the presence of brain involvement with or without systemic metastases when MBC was first documented. The OS, TTD, DFS, extracranial PFS and brain PFS were estimated by the Kaplan–Meier product limit method. The log-rank test was used to compare survival rates. A We identified 857 MBC patients who received palliative chemotherapy for the treatment of metastatic or recurrent breast cancer at Samsung Medical Center from 1999 to 2006. Two hundred sixty-four patients of the 857 patients were confirmed as HER2-overexpressing breast cancer patients. Among these patients, 140 patients were not treated with trastuzumab in pre-T period and 111 patients were treated with trastuzumab in post-T period. And remaining 13 patients were excluded from the analysis because of unavailability of their clinical data. Therefore, we included a total of 251 patients in this study .P=0.028 by χ2 test).The incidence of BM among the patients in this study was 30.7% (77 out of 251). The incidence of BM in each treatment group was 25.0% (35 out of 140) in the pre-T group and 37.8% (42 out of 111) in the post-T group .All 251 patients' characteristics are summarised in P=0.129). Leptomeningeal seeding had developed in three pre-T patients (8.6%) and nine post-T patients (21.4%). There was no significant difference between pre-T and post-T patients in terms of hormonal receptor status, distribution of initial TNM staging, pathological grade or performance status (PS). However, disease control rate of systemic extracranial lesions (CR+PR+SD) at the time of BM was much higher in the post-T group (75.6%) than in the pre-T group (48.1%) (P=0.020). The ‘only BM’ was more common in pre-T patients (14.3%) than in post-T patients (2.4%) (P=0.046). In addition, the development of the ‘first metastasis in brain’ was also more common in the pre-T group (25.7%) than in the post-T group (9.5%) (P=0.058).The patients' characteristics of 77 HER2-overexpressing MBC patients at the time of BM are summarised in Trastuzumab was administered to 27 of the 42 patients with BM (64.3%) as a first-line treatment with cytotoxic chemotherapy. Of the remaining 15 patients (35.7%), 10 were treated with trastuzumab as a second-line therapy, and three were treated with trastuzumab as a third- or more line therapy. There were 12 patients (28.6%) who had started trastuzumab after development of BM. Two patients were treated with trastuzumab continuously before and after BM. The median duration of trastuzumab treatment was 6.4 (1.2–22.5) months. Lapatinib was also administered in 10 patients (23.8%) for salvage treatment after BM.vs 14.3% in post-T, P=0.437). GKS was more common in the post-T group (59.5%) than in the pre-T group (28.6%) (P=0.007). WBRT was performed in 69 patients (89.6%) . Systemic chemotherapy was delivered to 45 patients (58.4%) and the median number of the systemic chemotherapy regimens was 1 (range 0–6). The patients who were treated with 1 or more systemic chemotherapy regimens were much more common in the post-T group than in the pre-T group . The median number of regimens delivered to patients was 0 (0–4) in pre-T patients and 2 (0–6) in post-T patients. The agents used in combination or as single systemic treatment regimens with or without trastuzumab after BM were anthracycline, taxane(s), gemcitabine, cisplatin, capecitabine and vinorelbine.Craniotomy was performed in nine patients (11.7%) (8.6% in pre-T P=0.001). 1-year survival rate was significantly higher in post-T group than in pre-T group . TTBM was longer in the post-T group  than in the pre-T group  with statistical significance (P=0.035 by log-rank test) (P=0.0005) (P=0.049), two or more systemic chemotherapy regimens after BM (P<0.0001), absence of ‘first metastasis in brain’ (P=0.045), extracranial disease control at the time of BM (P=0.0051) and 12 months or more extracranial PFS (P<0.0001) were identified as favourable prognostic factors for survival from BM (P=0.014 by Fisher's exact test) (The OS and TTD from development of systemic metastasis were 26.4 months (95% CI 20.9–31.9) and 9.6 months (95% CI 4.6–14.6), respectively. The OS from development of systemic metastasis was longer in post-T patients  than in pre-T patients  (nk test) . TTD fro=0.0005) . In a lo from BM . 11.9% oct test) .P=0.040, hazard ratio (HR) 5.069), two or more chemotherapy regimens after BM , ‘first metastasis in brain’ , extracranial disease control at the time of BM  and extracranial PFS ⩾12 month  were identified as independent prognostic factors for favourable survival from BM. Trastuzumab treatment after BM also showed a high HR, though it did not reach statistical significance .According to the results of univariate analysis, we performed Cox-regression multivariate analysis to identify independent factors for survival from BM. vs 25.0%, respectively, P=0.028). This finding can be explained by both the known aggressive nature and by the increased predilection to CNS metastases of HER2-positive tumours  is less common in the post-T group compared with the pre-T group  and TTD  that are independent of the trastuzumab line and duration  in addition to extracranial systemic disease control at the time of BM  . This finding confirms that an active systemic treatment has an important role and should be considered even in CNS metastases, because extracranial systemic control is a main contributing factor for predicting survival. It is commonly believed that most patients with BMs from advanced breast cancer do not die from cerebral progression, but rather from progression of systemic disease. This was apparently the major limiting factor in the survival of women before the trastuzumab period, and since the introduction of trastuzumab, the proportion of patients with controlled systemic disease dying from brain lesions has grown   . Only twas grown . ApparenP=0.014) . As showFurther, it is important to be very cautious when stopping trastuzumab treatment or switching to other drugs, because it is difficult to identify cases of trastuzumab resistance.P=0.040, HR 5.069) in this study  (is study , new theThe interpretations from this study have limitations. First of all, this is a retrospective single institutional study with a heterogeneous group of patients and treatments. However, this study population has some obstacles to develop a prospective randomised trial, such as relatively low prevalence, the urgent need for immediate supportive care and approval of the ethical problem to omit standard treatment. Taking allowance for these obstacles, this study has powerful value in providing guidelines for this particular clinical setting.In conclusion, the administration of trastuzumab in the treatment of HER2-positive breast cancer results in prolonged TTBM and TTD from BM, even after development of BM. Extracranial systemic disease control at the time of BMs and durable prolongation of extracranial systemic disease are the main independent prognostic factors for predicting survival with BM.


Despite of the recent success of EGFR inhibitory agents, the primary drug-resistant becomes a major challenge for EGFR inhibitor therapies. PTEN gene is an important positive regulatory factor for response to EGFR inhibitor therapy. Low-expression of PTEN is clearly one of the important reasons why tumor cells resisted to tyrosine kinase inhibitors.To investigate the drug-resistance reversal to gefitinb and the mechanism in PTEN low expression cells which radiated with X-rays in vitro, We demonstrated that H-157 lung cancer cells (low-expression of PTEN but phospho-EGFR overexpressed tumor cells) exposed to X-rays. The PTEN expressions and radiosensitizing effects of tyrosine kinase inhibitor before and after irradiation were observed. The cell-survival rates were evaluated by colony-forming assays. The cell apoptosis was investigated using FCM. The expressions of phospho-EGFR and PTEN were determined by Western blot analysis.The results showed that the PTEN expressions were significantly enhanced by X-rays. Moreover, the cell growth curve and survival curve were down-regulated in the gefitinib-treated groups after irradiation. Meanwhile, the radiation-induced apoptosis of tumor cells was increased by inhibition of the EGFR through up-regulation of PTEN.These results suggested that PTEN gene is an important regulator on TKI inhibition, and the resistance to tyrosine kinase inhibitors might be reversed by irradiation in PTEN low expression cancer cells. The EGFR is a receptor tyrosine kinase that regulates fundamental processes of cell growth and differentiation. Overexpression of EGFR and its ligands, were reported for various epithelial tumors in the 1980s  and geneCell culture media was provided by Tianjin Medical University Cancer Institute . Primary antibodies against phospho-EGFR and PTEN were obtained from Santa Cruz Biotechnology, Inc. ; Propidium Iodide (PI) and annexin V were purchased from Cell Signaling Company . Gefitinib was generously provided by AstraZeneca . All the other materials were from Cancer Institute of our university.The H-157 lung cancer cell line was kindly provided by Peking University Center for Human Disease Genomics. It was maintained in RPMI1640 supplemented with 20 mM HEPES (pH 7.4), 100 IU/mL penicillin, 100 mg/mL streptomycin, 4 mM glutamine and 10% heat-inactivated fetal bovine serum  in a humidified atmosphere of 95% air and 5% CO2 at 37°C.2) were irradiated by an X-ray generator with a 1.0-mm aluminum filter at 200 kVp and 10 mA, at a dose of 1.953 Gy/min, which was determined using Fricke's chemical dosimeter. Then they were incubated for another 48 h at 37°C. Addition of Gefitinib was carried out at the same time when the treatment of irradiation was performed. Radiation was performed in the Tianjin Medical University Cancer hospital.Exponentially growing H-157 cells in a tissue-culture flask , and incubated for 4 h at 37°C. After incubation, the MTT solution was carefully aspirated and replaced with 150 μL DMSO. Cell growth was analyzed on a plate reader by using SoftMax program . Experiments were performed in quadruplicate and repeated at least 3 times. At the same time, the antiproliferative effect of gefitinib on the growth profile in vitro of H-157 cell line was examined. Briefly, The cells were treated with different concentrations of gefitinib . The control and gefitinib-treated cells(1 × 104/well) were seeded on 6-well plates, and proliferation was evaluated by cell counting using a hemocytometer after 1,2,3,4,5,6 and 7 days of culture. All measurements were done in triplicate [Cell proliferation was determined by assessing the mitochondrial reduction of MTT. In brief, cells from the control and gefitinib-pre-treated groups were plated at 1 × 10iplicate ,26. The Clonogenic survival was the ability of cells to maintain clonogenic capacity and to form colonies. The treatment schedule for clone assay: there are 4 groups in the experiments . Cells in culture were irradiated with 1, 2, 4, 6, 8 and 10 Gy, and the gefitinib concentration was 100 nM. Briefly, after exposure to radiation, cells were trypsinized, counted, and seeded for colony formation in 60 mm dishes at 200 to 10000 cells/dish. After incubation intervals of 14 to 21 days, colonies were stained with crystal violet and manually counted. Colonies consisting of 50 cells or more were scored. Experiments were done in triplicate .To examine whether enhancement of apoptosis in X-ray irradiated H-157 cells overexpressed with PTEN was associated with gefitinib, we tested the effects of EGFR inhibitors on the enhancement of apoptosis in H-157 cells with and without irradiation. Cells from the irradiation and combined with Gefitinib groups (100 nM) were exposed to the same radiation dosages (6 Gy). At 48 h after irradiation, the cells were harvested. And then, cells were trypsinized, counted, and washed twice with cold PBS. Cells used for tests were stained with propidium iodide (PI) and annexin V for 15 min in the dark and analyzed by fluorescence-activated cell sorting (FACS) using Coulter EPICS and ModFit software . Each test was performed 3 times .Data was plotted as means ± standard deviation. Student's t test was used for comparisons. Differences were considered significant at P < 0.05.It was reported that H-157 cells might be overexpression of phospho-EGFR and low-expression of PTEN . In the As shown in Figure After different dosage radiation , the PTEN expression increased in a time-dependent manner. The highest expression were observed in H-157 cells treated with 4~6 Gy irradiation. At the same time, we also measured that PTEN expression increased at 3 h and returned to baseline at 12 h after irradiation  = D0 (irradiation+gefitinib group)/D0 (irradiation group) = 1.51/1.14 = 1.32. The SER in gefitinib-treated cells indicated that treatment with gefitinib significantly improved the biological effect of irradiation following PTEN high expressed. At the same time, the cell growth curve was also down-regulated by gefitinib after irradiation  pathway by removing the third phosphate from the inositol ring of the second messenger PIP3 . PTEN inAs the drug resistant cell line for tyrosine kinase inhibitors due to low expression level of PTEN protein , H-157 cIt must point out that cross talk between different pathways makes miscellaneous of signal transductions in tumor cells. Up to now, it is still unclear that whether there exist other factors of PI3K pathway producing influence for tyrosine kinase inhibitors efficacy except for PTEN protein. Whether the factors are influenced by irradiation, and then made some effect on the drug-resistance further remains unknown. These all need further research on PI3K pathway.Our study provides a new idea for drug-resistance reversal of TKI targeted therapy, and makes a beneficial exploration on tyrosine kinase inhibitors combined with radiotherapy in TKI resistant tumors. We believe that, with the further development of fundamental research, we are looking forward to an increasing application prospect of tyrosine kinase inhibitors in clinical practice.EGFR: epidermal growth factor receptor; PE: plating efficiency; SF: survival fraction; RBE: relative biological effect; D0; mean lethal dose; Dq: quasithreshold dose; N: extrapolation number; MTT: methyl thiazolyl tetrazolium; FCM: flowcytometry; TKI: tyrosine kinase inhibitor; PTEN: phosphatase and tensin homolog on chromosome ten; PIP3: phosphatidyinositol-3, 4, 5- triphosphate; PI3K: phosphatidylinositol-3-kinase.The authors declare that they have no competing interests.HZ wrote the paper. ZY designed the research. JW, LZ, and PW carried out the molecular genetics studies. CW carried out the data analysis. All authors have read and approved the manuscript.


Pantoea ananatis, a member of the Enterobacteriacea family, is a new and promising subject for biotechnological research. Over recent years, impressive progress in its application to L-glutamate production has been achieved. Nevertheless, genetic and biotechnological studies of Pantoea ananatis have been impeded because of the absence of genetic tools for rapid construction of direct mutations in this bacterium. The λ Red-recombineering technique previously developed in E. coli and used for gene inactivation in several other bacteria is a high-performance tool for rapid construction of precise genome modifications.P. ananatis was found to be highly toxic. A screening was performed to select mutants of P. ananatis that were resistant to the toxic affects of λ Red. A mutant strain, SC17(0) was identified that grew well under conditions of simultaneous expression of λ gam, bet, and exo genes. Using this strain, procedures for fast introduction of multiple rearrangements to the Pantoea ananatis genome based on the λ Red-dependent integration of the PCR-generated DNA fragments with as short as 40 bp flanking homologies have been demonstrated.In this study, the expression of λ Red genes in P. ananatis recipient strain. The procedure of electro-transformation with chromosomal DNA has been developed for transfer of the marked mutation between different P. ananatis strains. Combination of these techniques with λ Int/Xis-dependent excision of selective markers significantly accelerates basic research and construction of producing strains.The λ Red-recombineering technology was successfully used for rapid generation of chromosomal modifications in the specially selected  Pantoea ananatis belongs to the Enterobacteriacea family. The P. ananatis strain AJ13355 (SC17) was isolated from soil in Iwata-shi  as a bacterium able to grow at acidic pH and showing resistance to high concentrations of glutamic acid  based on the broad-host-range replicon of RSF1010 , has been constructed via substitution of CmR and B. subtilis sacB genes by the KmR gene from pUC4K [Another plasmid carrying λ Red genes and named pRSFRedkan [GenBank:om pUC4K .P. ananatis SC17 strain [Clones of FJ347163] plasmid lacking the exo gene encoding 5'→3' exonuclease.To establish which component of the λ Red system caused the toxic effect, we constructed the pRSFGamBet  plasmid was constructed by substitution of sacB and cat genes of pRSFGamBet for the KmR gene from pUC4K. This plasmid was introduced to the obtained SC17hisD::PlacUV5-sacB-cat strain. It is known from the literature [hisD locus in the P. ananatis chromosome is not known. Hence, the plasmid-carrier cells were independently electroporated with hisD-XhoI-1 and hisD-XhoI-2 ss-oligos complementary to each other. Cells were plated on LB medium containing 30% sucrose. No colonies were observed after 24-hour cultivation when hisD-XhoI-2 olig was used for electroporation. About 200 clones were obtained after electro-transformation with hisD-XhoI-1 olig. To test the phenotype of the obtained transformants, 100 clones were replicated on the following solid mediums: LB medium supplemented with 30% sucrose, LB medium supplemented with Cm and M9 minimal medium. Seven of the tested clones had SucRCmSHis+ phenotype. These colonies were further verified by PCR and restriction of the amplified product. The presence of the expected mutation (substitution of the SmaI by XhoI recognition site) was confirmed in all of the seven SucRCmSHis+ clones.The terature ,42 that P. ananatis.Although the frequency of selection of the desired mutation was not high, in principle, λ Beta-dependent integration of ssDNAs allowed the construction of the unmarked mutations in the E. coli cells by the λ Red functions [P. ananatis, a bacterium of interest in the field of metabolic engineering. A broad-host-range λ Red-expressing plasmid useful for P. ananatis was constructed. The observed general toxicity of simultaneous expression of the λ gam, bet, and exo genes for P. ananatis SC17 cells was overcome by selection of the special recipient, SC17(0).It is currently well established that high-frequency recombination between short homologies can be catalyzed in unctions -7,43-45.E. coli cells can lead to toxic effects [E. coli in detail. In addition to its direct influence on traditional recombination pathways due to inhibition of RecBCD and SbcCD nucleases by λ Gam, the activity of λ Red proteins can interfere with the processes of replication and repair  lower intracellular level of λ Red proteins in the mutant cell, caused by reduced level in the synthesis of λ Red proteins or by increased efficiency of the specific proteolysis of λ Red proteins, (ii) decreased affinity of specific targets for interaction with λ Red proteins or increased level of biosynthesis of these targets. As for the P. ananatis mutant strain obtained, both variants of the explanation may be possible. As mentioned in the Results section, it is not very probable that SC17(0) strain possesses reduced level of the synthesis of λ Red proteins. But this possibility can not be completely rejected.Toxicity of expression of λ Red genes for the bacteria closely related to P. ananatis chtomosome. All types of chromosome modifications constructed in E. coli by the λ Red-mediated recombineering, have been successfully reproduced in SC17(0) with pRSFRedTER as λ Red-expressing plasmid. Typically, the yield of recombinants varied from several tens to several hundreds per trial.Nevertheless, even without information concerning the nature of the SC17(0) mutation, it is possible to use the corresponding strain for the desired λ Red-mediated rearrangements of P. ananatis strains of interest using the method of electro-transformation with chromosomal DNA. This method is a unique way to combine the set of marked mutations constructed in different P. ananatis strains into a single strain. In addition, potentially, co-transfer of rather closed mutations could be prevented by digestion of the chromosomal DNA by the appropriate restriction endonucleases, whose recognition sites are located between the mutations.Using SC17(0) as the initial recipient for λ Red-promoting modifications it is subsequently possible to transfer the marked mutation to other lox, Flp/FRT) are well-known [int and xis genes that, together with the host factors , provide the following reaction: attLλx attRλ = attPλ+attBλ. Peredelchuk & Bennet [attLλ and attRλ sites. The attBλ site remaining in the chromosome after marker excision cannot recombine with attLλor attRλ site in the next steps of strain construction. Thus, repeated action of the system would not influence the strain stability. Certainly, residual attBλ sites could recombine with each other via host general recombination system or provoke replication errors, especially if many left over scars (attBλ) are presented in chromosome and their positions were rather close to each other. Such events would lead to deletions of chromosomal fragments.As the number of combined mutations of interest exceeds the number of available antibiotic resistance markers, curing of the intermediate strains from the used selective markers is necessary. Moreover, the presence of antibiotic resistance genes in the genomes of the industrial strains is rigorously restricted by the legislations of different countries. A wide variety of systems for marker curing based on the site-specific recombination systems  or ssDNA in any other P. ananatis strain carrying the dual selective/counter-selective marker in the target point of the genome.Finally, the two-step procedure for introducing the unmarked mutations into the P. ananatis genes whose products were involved in central metabolism, respiration, transcription regulation, etc. Several marker-less P. ananatis strains carrying multiple (> 10) different chromosomal modifications including "in-frame" deletions, point mutations, rearrangements of regulatory regions, in particular, were constructed for basic research and applied purposes using combined application of the λ Red-Int/Xis systems and electro-transformation with chromosomal DNA.Up to the present, the developed λ Red-mediated method has been used for deletion of more than 50 P. ananatis. In combination with the established procedures of λ Int/Xis-dependent marker elimination and electro-transformation with chromosomal DNA, this method provides a simple route to obtaining marker-less strains carrying multiple mutations of different types . The described approach of selection of the recipient strain resistant to expression of λ Red genes could be useful in exploiting λ Red-recombineering in other bacteria.The λ Red-mediated recombineering has been adjusted for rapid and efficient construction of genome rearrangements in Strains and plasmids used or generated in this study are listed in Table E. coli and P. ananatis strains were cultivated with aeration in LB medium at 37°C and 34°C, respectively. The following antibiotic concentrations were used to select transformants and to maintain the plasmids: Km – 40 mg/l, Cm – 50 mg/l. The M9 salt medium supplemented with galactose (1 g/l) or glucose (1 g/l) was used to select Gal+ or His+ cells.DNA manipulations were performed according to standard methods . RestricE. coli galK and P. ananatis hisD genes, a removable KmR marker flanked by attLλ and attRλ was amplified with galK-5/galK-3 and hisD-5/hisD-3 primers, respectively. The pMW-attLλ-KmR-attRλ plasmid was used as DNA template. To obtain insertion into the P. ananatis hisD gene, the Plac-sacB-cat cassette was amplified in PCR with his-Plac-5/his-cat-3 primers using pRSFPlacsacB plasmid as template.To provide cassettes for λ Red-dependent integration, the appropriate selective marker was amplified by PCR with oligos containing on their 5'-ends 36-nt sequences homologous to the target region. To disrupt XhoI restriction site in the center and arms homologous to the sequence surrounding native SmaI site of the P. ananatis hisD. As a result, the short dsDNA fragment containing the XhoI recognition site in its center and 33 bp long arms homologous to the target region, was obtained. The obtained fragment was amplified and extended by PCR with the primers his-SL and his-SR. The resultant DNA fragment generated by PCR contained XhoI recognition site in its center flanked with 82 bp arms homologous to the appropriate site in P. ananatis hisD gene.First, his-XhoI-1 and his-XhoI-2 oligos complementary to each other were annealed. Both oligos contained sequences corresponding to the P. ananatis strain grown at 34°C with aeration was diluted with fresh LB broth 100 times and the cultivation was continued up to the OD600 = 0.5–0.8. Cells from ten millilitres were washed three times with an equal volume of deionized ice water followed by washing with 1 ml of 10% cold glycerol and resuspended in 35 μl of 10% cold glycerol. Just before electroporation, 10–100 ng of the plasmid DNA dissolved in 2 μl of deionized water was added to the cell suspension. The procedure of plasmid electro-transformation was performed using the GenePulser and Pulse Controller . The applied pulse parameters were: electric field strength of 20 kV/cm, time constant of 5 msec. After electroporation, 1 ml of LB medium enriched with glucose (5 g/l) was immediately added to the cell suspension. Then the cells were cultivated under aeration at 34°C for 2 h and plated on LB-agar containing the appropriate antibiotic. This was followed by an overnight incubation at 34°C. A competence of P. ananatis cells, determined for RSF1010 plasmid, was 106 CFU per μg of DNA. Typically, 105–106 antibiotic resistant colonies were obtained per 108 survivors following electroporation.An overnight culture of P. ananatis or E. coli strains harbouring the plasmid, expressing appropriate λ Red genes, grown in LB broth with Cm  or Km  were diluted 100 times with the same fresh medium supplemented with 1 mM IPTG for induction of the λ Red genes. At culture density of 0.5–0.6, electro-competent cells were prepared as described above. From 200 to 500 ng of a PCR-generated linear dsDNA or 100 ng of a ss-oligos were used for transformation. Electroporation was carried out at electric field strength of 25 kV/cm and time constant of 5 msec for both types of DNA substrates. The chromosome structure of the obtained transformants was verified in PCR with galK-t1/galK-t2 primers for E. coli galK gene disruption, hisD-t1/hisD-t2 for P. ananatis hisD gene disruption and for insertion of the double selective/contra-selective marker into the P. ananatis hisD.Overnight cultures of Gene disruption provided with pKD46 plasmid was performed as described in (4).600 = 0.8–1.0. Electro-competent cells were prepared as described above. From 1 to 2 mg of a chromosomal DNA, isolated using a Genomic DNA Isolation Kit (Sigma), was used for transformation. Electroporation was carried out with an electric field strength of 12.5 kV/cm and time constant of 10 msec.Cells were grown in LB medium up to ODSDS-PAGE of cell extracts was performed according to Laemmli  with polR: ampicillin resistance; Cm: chloramphenicol; CmR: chloramphenicol resistance; cat: chloramphenicol resistance gene; Km: kanamycin; KmR: kanamycin resistance; kan: kanamycin resistance gene; sacB: gene encoding the levansucrase; attLλ: attL site of phage λ; attRλ: attR site of phage λ; attBλ: attB site of phage λ; IPTG: isopropyl-β-D-thiogalactopyranoside; nt: nucleotide; bp: base pair(s); oligos: oligonucleotides; PCR: polymerase chain reaction; ssDNA: single-stranded DNA; dsDNA: double-stranded DNA.Ap: ampicillin; ApP. ananatis strains differed from SC17(0). LIG constructed all recombinant plasmids, expressing λ Red genes, tested their function initially in E. coli, and edited the manuscript. TMK performed the λ Red-driven modifications of the chromosome of P. ananatis SC17(0) strain. IGA performed λ Red-driven oligos integration into chromosome of P. ananatis SC17 strain. SVM supervised and coordinated the work and edited the manuscript. All authors have read and approved the final version of the manuscript.JIK and YH are the project leaders in AGRI and in Ajinomoto, respectively. JIK obtained SC17(0), designed the main experiments concerned with λ Red-driven modifications, and drafted the manuscript. YH developed the transfer of λ Red-driven mutations to Plasmids constructed for this study. Detailed description of plasmids constructed in this study.Click here for filePrimers used for this study. List of primers.Click here for file


Staphylococcus aureus and Pseudomonas aeruginosa are often found together in the airways of cystic fibrosis (CF) patients. It was previously shown that the P. aeruginosa exoproduct 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) suppresses the growth of S. aureus and provokes the emergence of small-colony variants (SCVs). The presence of S. aureus SCVs as well as biofilms have both been associated with chronic infections in CF.S. aureus to form a biofilm in association with the formation of SCVs. The emergence of SCVs and biofilm production under HQNO exposure was shown to be dependent on the activity of the stress- and colonization-related alternative sigma factor B (SigB). Analysis of gene expression revealed that exposure of a prototypical S. aureus strain to HQNO activates SigB, which was leading to an increase in the expression of the fibronectin-binding protein A and the biofilm-associated sarA genes. Conversely, the quorum sensing accessory gene regulator (agr) system and the α-hemolysin gene were repressed by HQNO. Experiments using culture supernatants from P. aeruginosa PAO1 and a double chamber co-culture model confirmed that P. aeruginosa stimulates biofilm formation and activates SigB in a S. aureus strain isolated from a CF patient. Furthermore, the supernatant from P. aeruginosa mutants unable to produce HQNO induced the production of biofilms by S. aureus to a lesser extent than the wild-type strain only in a S. aureus SigB-functional background.We demonstrated that HQNO stimulates S. aureus responds to HQNO from P. aeruginosa by forming SCVs and biofilms through SigB activation, a phenomenon that may contribute to the establishment of chronic infections in CF patients.These results suggest that  Staphylococcus aureus and Haemophilus influenzae, but these organisms are usually outnumbered by Pseudomonas aeruginosa as patients become older. However, S. aureus often persists in the airways of CF patients and the role of S. aureus in the progression of CF patients to respiratory failure is not yet understood whereas infections with P. aeruginosa is considered as one of the main factors for a decline in lung function and mortality [Although cystic fibrosis (CF) is fundamentally a genetic disorder, the majority of patients with CF may ultimately succumb to respiratory failure subsequent to chronic bacterial infections . In earlortality . Interesortality ,3.Infections with mixed microbial communities are common, although very little is known about the importance and the impact of interspecies interactions . It is nS. aureus with physiological concentrations of the P. aeruginosa exoproduct 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) selects for a sub-population of slow-growing respiratory deficient S. aureus named small-colony variants (SCVs) [S. aureus against P. aeruginosa [S. aureus SCVs are often isolated from chronic infections [S. aureus SCVs possess an increased capacity to invade and persist in host cells [S. aureus infections.It has recently been shown that prolonged growth of s (SCVs) . Furtherruginosa . S. aurefections , as in tfections -16. Sevest cells ,15,17, wst cells . Using Sst cells . Furtherst cells , which ssarA  [agr (accessory gene regulator) system [agr system controls the transition from colonization to dissemination by up-regulating the expression of several exotoxins and proteolytic enzymes and by repressing the expression of cell-surface proteins involved in colonization [agr [S. aureus.SigB is involved in the response to environmental stresses such as during stationary phase, heat exposure and change in osmotic pressure . Moreovegulator) ,23. SarA) system . In turnnization . agr [26ion [agr ,28 and Sion [agr  are knowS. aureus [ica gene cluster, whereas the second mechanism is ica-independent. With opposite effects, SarA and agr are both involved in the ica-independent mechanism of biofilm formation. SarA is thought to be indirectly required for the initial attachment step to biological matrices [agr is controlling the dispersal process of biofilms [et al. [ica-independent biofilm formation and suggested that SigB acts upstream of the agr system, allowing the formation of biofilm to be regulated as a function of environmental factors. Noteworthy, biofilms have been linked to chronic infections, especially in the case of those found in the airways of CF patients [At least two different mechanisms of biofilm formation exist in . aureus ,29-33. Tmatrices ,33, whilbiofilms . Recentl [et al.  have shopatients ,34, and patients ,35.S. aureus when exposed to P. aeruginosa HQNO.The aim of this study was to investigate the association between the activity of SigB, the emergence of SCVs and biofilm production in S. aureus and causes the emergence of SCVs. Isolates CF1A-L and CF1D-S are two related strains co-isolated from a CF patient which have a normal and a SCV phenotype, respectively (see Methods). At a concentration of 10 μg/ml, HQNO significantly attenuated the growth of CF1A-L  whereas HQNO had no apparent effect on the growth of CF1D-S which was already significantly slower than that of CF1A-L in the absence of HQNO  . These results corroborate that HQNO generally suppresses the growth of normal S. aureus populations and provokes the emergence of SCVs from strains of different origins.Fig. st) Fig. . Hence, S. aureus. Fig. hemB (which is a stable laboratory-derived SCV) ensured that SCVs (and not revertants) are indeed responsible for this increase in biofilm production (at least in the case of NewbouldhemB). Furthermore, as shown in Mitchell et al. [Several pairs of related normal and SCVs strains were used in order to study the effect of HQNO on biofilm production by l et al. , supplemhemB, CF03-S, CF07-S and CF1D-S  for strain Newman, CF03-L and CF1A-L even after sub-culturing strains in HQNO-free medium (data not shown). This indicates that an exposure of S. aureus to HQNO may result in a sustained increase in biofilm production. Overall, these results suggest that HQNO increases biofilm production in normal S. aureus strains and that the sustained effect of HQNO on biofilm production in subsequent HQNO-free medium may result from the relative increase in the sub-population of SCVs which are good biofilm producers.Besides, the presence of HQNO at 10 μg/ml did stimulate biofilm production in the normal strains Fig. . This obD-S Fig. . MoreoveD-S Fig. . InteressigB were used to determine whether SigB is involved in the emergence of SCVs and biofilm production under an exposure to HQNO. Fig. sigB+ background. HQNO significantly increased the presence of SCVs in strain Newbould, but not in NewbouldΔsigB . However, the ability of HQNO to increase biofilm formation was observed with strain Newbould, but not with NewbouldΔsigB , hla, sarA and gyrB at the exponential growth phase for strains Newbould and NewbouldΔsigB exposed or not to HQNO. The expression of asp23 and fnbA was evaluated in order to verify the hypothesis that SigB is activated during HQNO exposure. The gene asp23 is a well-known marker for SigB activity as for the gene fnbA, although the transcription of the latter is not exclusively influenced by SigB [asp23 and fnbA expression. The effect of HQNO on the expression of asp23 and fnbA was further confirmed with the sequenced strain Newman (data not shown). These results suggest that SigB activity is increased by HQNO.Fig.  by SigB ,19,22,37agr system is known to be reduced in SCVs [agr quorum-sensing system due to the general suppression of growth toward normal strains (likely mediated through the inhibition of the electron transport chain by HQNO [sigB to HQNO significantly repressed the expression of hld (the effector of the agr system).The activity of the  in SCVs ,38-41. W by HQNO ) but alsagr observed under exposure to HQNO, it was also justified to measure the expression of the α-hemolysin gene hla which can be influenced by both agr and SigB [hla was only significantly repressed in Newbould and not in NewbouldΔsigB by the presence of HQNO  as well as on the expression of sarA (up by a SigB-dependent mechanism).Overall, these results suggest that exposure of  in SCVs ,19,20,41S. aureus is representative of what may happen when P. aeruginosa and S. aureus are in close proximity during a co-infection, we conducted experiments in which S. aureus was exposed to supernatants from overnight cultures of P. aeruginosa as well as experiments using a double chamber co-culture model. We used the E. coli strain K12 in control experiments to ensure that the observed effect was specific to P. aeruginosa and was not only caused by the close proximity of a Gram-negative bacterium or non specific alterations of the growth medium. We used E. coli because it is known that this bacterium does not produce HQNO . Fig.P. aeruginosa PAO1 inhibits the growth of the S. aureus strain CF1A-L whereas this phenomenon was not observed with E. coli K12. The supernatant collected from an overnight culture of PAO1 significantly inhibited the growth of S. aureus. This growth inhibition was accompanied by a significant increase in biofilm production  at all [pqsL mutant is specifically altered in HQNO biosynthesis [pqsA and pqsL mutants in order to distinguish the global impact of all P. aeruginosa HAQs from the specific impact of HQNO on biofilm production by S. aureus. Fig. pqsA and pqsL mutants is not impaired compared to that of the parental strain PA14, thus excluding variations in supernatant composition caused by differences in growth rates among strains. Fig. P. aeruginosa PA14 stimulates biofilm production by S. aureus CF1A-L in comparison to the supernatant from the pqsL mutant (specific HQNO-minus strain). The effect of different doses of supernatants from overnight cultures of P. aeruginosa PA14, the pqsA mutant, the pqsL mutant or E. coli K12 on biofilm production by S. aureus CF1A-L is shown in Fig. pqsA mutant  than the pqsL mutant. This result can be explained by the fact that other HAQs secreted by P. aeruginosa, although less potent than HQNO, can also have a growth-inhibitory activity against S. aureus [P. aeruginosa stimulated biofilm production in comparison to E. coli, suggesting that other P. aeruginosa exoproducts can indeed stimulate biofilm production by S. aureus.We used the ) at all ,45, wherynthesis . Thus, w. aureus . NoteworS. aureus Newbould is in contact with the supernatant from PA14 is significantly higher than that seen with supernatants from the pqsA and pqsL mutants. Surprisingly, both mutants did not significantly stimulate biofilm production by Newbould as that observed for CF1A-L, suggesting that differences between S. aureus strains may exist in respect to their response to the presence of non-HQNO exoproducts. As expected, biofilm production by NewbouldΔsigB in contact with supernatants from the three P. aeruginosa strains was significantly inferior to that observed using the PA14 supernatants with strain Newbould. Moreover, supernatants from PA14 generally did not significantly stimulate biofilm production by NewbouldΔsigB in comparison to supernatants from pqsA and pqsL mutants, which confirms that SigB is involved in HQNO-mediated S. aureus biofilm production. Overall, the results of this section support the hypothesis that HQNO from P. aeruginosa stimulates biofilm production by S. aureus through a SigB-dependent mechanism.Fig. P. aeruginosa exoproduct HQNO increases the production of biofilm by S. aureus. The effects on biofilm production, as well as on growth, were only seen on normal strains whereas the already high biofilm formation and slow growth rate of SCVs were not altered by the presence of HQNO. It is known that HQNO specifically inhibits electron transport in Gram-positive bacteria [S. aureus. We indeed found that a pre-treatement of S. aureus with HQNO still led to a subsequent increase in biofilm formation even after HQNO removal. This sustained effect is probably associated with the increased proportion of the sub-population of SCVs resulting from HQNO exposure. An exposure of S. aureus to HQNO may thus, in addition to its immediate effect, favor the emergence of SCVs having a long-term impact on biofilm formation.We found that the bacteria  and its S. aureus SCVs was previously observed by Hoffmann et al. [S. aureus and P. aeruginosa further increases the formation of biofilm by S. aureus. Besides, it is well known that the abnormal function of the CF transmembrane conductance regulator (CFTR) protein in CF patients has profound consequences on the airway physiology and it will be of great interest to determine whether other parameters related to the CF airways influence the emergence of SCVs and the production of biofilms by S. aureus. The expression of virulence factors in S. aureus is indeed controlled by diverse and complex regulatory networks in a time- and environment-dependent manner, being influenced for example by ionic forces, pH and O2 [S. aureus specifically responds to the particular environment of CF airways. Whether this response is SigB-dependent and will lead to the emergence of SCVs and biofilm production remains to be determined.Aminoglycosides are also known to favor the emergence of SCVs  and are n et al. . It is tH and O2 . ConsequS. aureus have been previously reported [sigB will importantly influence the outcome of the HQNO-mediated interaction between P. aeruginosa and S. aureus. We are currently studying S. aureus isolates from CF patients co-infected with P. aeruginosa which are not influenced by the presence of P. aeruginosa. This, in addition to the observation that differences between S. aureus strains exist relative to their response to HAQs  involved in adhesion to host tissues, and in the repression of several exotoxins  and proteolytic enzymes [S. aureus from disseminating and will rather favor tissue colonization, biofilm production and invasion of host cells. It has indeed been suggested that S. aureus FnBPs mediates cellular invasion [hla [S. aureus to HQNO up-regulates the expression of fnbA and represses the expression of hla. However, whether or not HQNO and P. aeruginosa increase the invasion of host cells by S. aureus remains to be confirmed. Interestingly, O'Neil et al. [ica-independent mechanism of biofilm formation. It is thus possible that FnBPs are directly responsible for the observed HQNO-mediated SigB-dependent increase in biofilm production and, more specifically, FnBPA which is under the control of SigB for expression [S. aureus SCVs.The effect of HQNO on the regulators SarA,  enzymes ,25. It iinvasion ,54 whereion [hla . Accordil et al.  have recpression ,19,22,37S. aureus during polymicrobial infections. Our results and those of Machan et al. [S. aureus, although not as efficiently as HQNO. Moreover, it is known that other P. aeruginosa exoproducts such as pyocyanin have an inhibitory activity against the electron transport chain of S. aureus [pqsA-E genes [pqsA and pqsL mutants we have observed on the growth (data not shown) and biofilm formation of S. aureus  broth . The use of BHI to study our SCV strains as well as in the experiments involving quantification of SCVs is validated in the Additional file Pseudomonas aeruginosa PAO1 [pqsA and pqsL mutants [Escherichia coli K12 were grown in trypticase soy broth (TSB) .The relevant characteristics of the strains used in this study are shown in Table cassette ,17, wereosa PAO1 , PA14 [6osa PAO1 , the PA1 mutants ,46 and Eet al. [The relatedness of each of the co-isolated strains within the pairs CF03-L/CF03-S, CF07-L/CF07-S and CF1A-L/CF1D-S was confirmed by MVLA as described by Sabat et al. . The strS. aureus overnight cultures were used at an A595 nm of 0.1 to inoculate BHI broths supplemented or not with 10 μg/ml of HQNO . Cultures were then incubated at 35°C/225 RPM and samples were taken at different time points for determination of CFU by spreading 10-fold dilutions on trypticase soy agar (TSA) plates . Plates were incubated at 35°C for 24 and 48 h for normal and SCV strains, respectively. For the growth curves of P. aeruginosa PA14 and the pqsA and pqsL mutants, overnight cultures were used to inoculate TSB. Cultures were then incubated at 35°C/225 RPM and samples were taken at specified time points in order to evaluate their turbidity at A595 nm.A595 nm of 2.0 in PBS at 4°C. Determination of SCV CFUs was done by serial dilution plating. SCV counts were obtained by plating on TSA containing gentamicin  at 4 μg/ml followed by an incubation of 48 h at 35°C. As shown in Additional file We have quantified SCVs by taking advantage of their reduced susceptibility to aminoglycosides as described elsewhere with few modifications ,64,65. AThe minimal inhibitory concentrations (MICs) of gentamicin for all strains were determined by a broth microdilution technique, following the recommendations of the Clinical and Laboratory Standards Institute (CLSI) guidelines , except hemB in proximity of a well loaded with hemin as an example of a positive auxotrophism result.In the context of SCVs, auxotrophism is defined as the requirement of specific compounds in order to regain a normal growth phenotype . An agarP. aeruginosa PAO1 [Overnight cultures were used to inoculate TSB at a dilution of 1:100. Cultures were then incubated 20 h at 35°C/225 RPM before collecting the culture supernatants by centrifugation. Similar culture conditions were previously shown to allow maximal production of HQNO by osa PAO1 . The supS. aureus, three colonies grown on blood agar plates were used to inoculate BHI broths containing 0.25% glucose with or without 10 μg/ml of HQNO and cultures were incubated for 18 h. These cultures were used to adjust an appropriate volume of BHI-0.25% glucose to 0.5 Mcfarland for transfer into wells of a flat-bottom polystyrene microtiter plate containing half volume of the same medium with or without HQNO . For experiments evaluating the effect of culture supernatants from P. aeruginosa and E. coli on S. aureus biofilm production, a S. aureus 0.5 Mcfarland suspension was prepared in BHI-0.5% glucose and transferred into wells of a microtiter plate containing half the volume of the supernatant to be tested. The plates were incubated at 35°C for 48 h. The supernatant was then discarded and the wells were delicately washed three times with 200 μl of PBS. The plates were dried, stained for 30 min with crystal violet, washed twice with 200 μl of water and allowed to dry again. A volume of 200 μl of 95% ethanol was added to each well and plates were incubated at room temperature for 1 h with frequent agitation. The absorbance of each well was then measured at 560 nm using a plate reader (Bio-Tek Instruments). The biofilm formation of each culture tested was evaluated in four replicates. The A560 nm values  representing the biofilm production for each of the strains used in Fig. For studying the effect of HQNO on biofilm production by S. aureus gene expression, overnight cultures were used to inoculate broth at an A595 nm of 0.1. Bacteria were then grown until the unexposed control culture reached an A595 nmbetween 0.9 and 1.0. Bacteria were collected and treated with RNAprotect . RNA was extracted from the cell pellets after treatment with lysostaphin (Sigma-Aldrich)  using the RNeasy Mini kit and the RNase-free DNase set (QIAGEN). A second DNase treatment was also done with the DNA-free kit . One μg of total RNA was reverse transcribed with 0.5 mM deoxynucleotide phosphate, 50 ng of random hexamers and 200 U of Invitrogen Superscript II reverse transcriptase, according to the manufacturer's recommendations . RNA was hydrolyzed and the cDNAs were purified with the QIAquick PCR purification kit (QIAGEN). One microliter of the cDNA preparation was amplified on the Stratagene MX3000P Real-Time PCR instrument with the Jump Start Taq DNA polymerase (Sigma-Aldrich), SYBR Green and 100 nM of the following primers:In order to evaluate the effect of HQNO (10 μg/ml) on asp23-RT-FWD 5'-TCGCTGCACGTGAAGTTAAA-3',asp23-RT-REV 5'-CAGCAGCTTGTTTTTCACCA-3',fnbA268-RT-FWD 5'-ACAAGTTGAAGTGGCACAGCC-3',fnbA341-RT-REV 5'-CCGCTACATCTGCTGATCTTGTC-3',hld-RT-FWD 5'-TAATTAAGGAAGGAGTGATTTCAATG-3'hld-RT-REV 5'-TTTTTAGTGAATTTGTTCACTGTGTC-3'hla-RT-FWD 5'-AATGAATCCTGTCGCTAATGCCGC-3'hla-RT-REV 5'-CTGAAGGCCAGGCTAAACCACTTT-3'sarA-RT-FWD 5'-CAAACAACCACAAGTTGTTAAAGC-3'sarA-RT-REV 5'-TGTTTGCTTCAGTGATTCGTTT-3'16SrRNA-RT-FWD 5'- TCGTTTAACACGTTTAGGTTCA-3',16SrRNA-RT-REV 5'- GAACTGTATCAGTTGGTTTCGCAC-3',gyrB-RT-FWD 5'-GGTGCTGGGCAAATACAAGT-3',gyrB-RT-REV 5'-TCCCACACTAAATGGTGCAA-3'.t) of the 16S RNA or gyrB of each condition as the calibrator .Reaction mixtures were denatured for 10 min at 95°C, followed by 35 cycles of 30 s at 95°C, 1 min at 60°C and 1 min 30 s at 72°C. Dissociation and standard curves were obtained to insure the specificity and the efficiency of reactions. cDNA synthesis reactions without reverse transcriptase were also routinely carried out. The relative expression ratios were calculated by using the cycle threshold  or top (S. aureus) chambers of 0.4-μm pore polycarbonate membrane inserts . S. aureus was inoculated at an 595 nm Aof 0.01, whereas P. aeruginosa or E. coli were inoculated at an 595 nm Aof 0.1. The cultures were incubated at 35°C/80 RPM for 6 h and samples were taken for SCV enumeration and total CFU counts as well as for RNA extraction. No bacterial cross-contamination was detected by culture plating up to at least 9 h of incubation.Overnight cultures of t-tests were used when only two conditions were compared. Two-way ANOVA with Bonferroni's post tests were used to compare the response of different strains and/or different conditions as a function of the concentration of HQNO or bacterial culture supernatants. Statistical analyses of qPCR data were done on mean ΔCt. CFU counts or SCV frequencies were transformed in based-10 logarithm values before being used for statistical analyses that were carried out with the GraphPad Prism Software (v.5.00). Statistical tests used for the analysis of each experiment are specified in figure legends.One-way analysis of variance followed by Dunnett's multiple comparisons test or Tukey's multiple comparisons test were used when several conditions or strains were compared at the same time whereas unpaired GM, DLS and AEA carried out the experiments. GM, DLS, ED, AMC, EHF, SM and FM designed and conceived the study. GM and FM wrote the paper. All authors read and approved the final manuscript.Validation of the use of BHI as the growth medium to induce and study SCVs. (A) Growth curves expressed in absorbance at 595 nm for the strains Newbould, NewbouldhemB, CF07-L and CF07-S. The growth of NewbouldhemB and CF07-S was supplemented or not with 5 μg/ml of hemin and 1 μg/ml of menadione, respectively. Results show that SCVs present their slow-growth phenotype in BHI unless supplemental hemin or menadione is added to the broth. (B) Pictures of colonies from strains Newbould, NewbouldhemB, CF07-L and CF07-S grown on BHI agar for 16 hours. Results show that SCVs retain their slow-growth phenotype on BHIA in comparison to normal strains. (C) Appearance of the colonies obtained from the cultures shown in A at the 12-h time point and plated on Mueller-Hinton agar (MHA) for 36 hours. Results show that normal strains grow efficiently on MHA whereas SCVs taken from BHI broth (cultures shown in A and supplemented or not with hemin or menadione) still present their slow-growth phenotype once plated back on MHA.Click here for fileMinimal inhibitory concentrations (MICs) of gentamicin for the studied strains. Results of this file show that MICs of gentamicin for SCVs are of 8 μg/ml whereas those of normal strains are below 2 μg/ml.Click here for fileAppearance of HQNO-induced SCVs selected on gentamicin-containing agar and streaked back on TSA plates. Pictures are showing CF07-L, CF07-S and HQNO-induced SCVs selected on gentamicin-containing agar and streaked back on TSA plates. The bottom pictures show streaks of three isolated SCVs on TSA plates. Many more SCVs were similarly tested and our results showed that at least 85% of the SCVs isolated from gentamicin plates were keeping their slow-growth phenotype when subsequently grown on TSA without gentamicin.Click here for fileAuxotrophism found among HQNO-induced SCVs. Auxotrophism found among HQNO-induced SCVs generated from the normal cystic fibrosis strains CF07-L and CF1A-L.Click here for filehemB in proximity of a well loaded with heminGrowth of Newbould. Growth of NewbouldhemB in proximity of a well loaded with hemin as an example of a positive auxotrophism result. The auxotrophism of NewbouldhemB for hemin is seen by observing normal growth only within the diffusion zone of a well loaded with hemin.Click here for fileNon-normalized absorbance values at 560 nm representing biofilm production for each of the strains used in Fig. Click here for file